Figure 7.

A: Evaluation of early apoptosis, measured as percentage of cells positive for annexin V by FACS analysis. Cells and spheroids were incubated with 3 μMol/L doxorubicin for 18 h, in the absence (white columns) or in the presence (hatched columns) of 5 μMol/L YC-1 for 24 h (6 h alone and 18 h together with doxorubicin). Apoptosis was then measured in MCF7 cell monolayers under normoxic conditions (Monolayer Normoxia), MCF7 3-D spheroids (Spheroids), and MCF7 cell monolayers under hypoxic conditions (Monolayer Hypoxia). ***, P < 0.0001 versus Monolayer Normoxia without YC-1; **, P < 0.01 versus Monolayer Normoxia without YC-1; °°, P < 0.001 versus Spheroids without YC-1; °, P < 0.05 versus Monolayer Hypoxia without YC-1. The figures are representative of three similar experiments, performed in duplicate. B: Evaluation of apoptosis by detection of caspase activity by fluorogenic assays measured as amount of cleaved fluorogenic substrates from cellular lysates by spectrophotometric analysis. Cleavage of the synthetic fluorogenic substrate Ac-LEDH-AMC by caspase-9. Cells and spheroids were incubated with 3 μMol/L doxorubicin for 18 h, in the absence (white columns) or in the presence (hatched columns) of 5 μMol/L YC-1 for 24 h (6 h alone and 18 h together with doxorubicin). Apoptosis was then measured in MCF7 cell monolayers under normoxic conditions (Monolayer Normoxia), MCF7 3-D spheroids (Spheroids), and MCF7 cell monolayers under hypoxic conditions (Monolayer Hypoxia). ***, P < 0.0001 versus Monolayer Normoxia without YC-1; **, P < 0.0001 versus Monolayer Normoxia without YC-1; °°, P < 0.001 versus Spheroids without YC-1; °, P < 0.001 versus Monolayer Hypoxia without YC-1. The figures are representative of three similar experiments, performed in duplicate.